DESCRIPTION (from applicant's abstract): Three NO synthase isoforms function differently in human health and disease. The proposed goal is to define the mechanism of NO synthesis and the protein structural elements that control activity in all three NOS. The biochemical and crystallographic work suggests mechanisms and identifies structural elements that may control formation of the active NOS dimer, the function of the essential cofactor tetrahydrobiopterin and heme in NO synthesis, and electron transfer. The proposed aims propose a variety of biochemical, kinetic, molecular biological, and biophysical studies to examine roles for these structural elements and establish mechanisms in all three NOS. This will improve the understanding of NOS structure-function and how tetrahydrobiopterin (H4B) and heme participate in NO synthesis. Aim I. Evaluate and establish mechanisms for N-terminal protein elements in active dimer formation. NOS contain an N-terminal b-hairpin hook that is followed by a CxxxxC motif that can either bind metal ions or form a disulfide link between subunits. The investigator hypothesizes that active dimer formation requires the N-terminal hooks to bind to their partner subunit, and the swapping is controlled by the CxxxC motif. The principal investigator will use mutagenesis to study how hook residues and the CxxxxC motif control hook swapping and active dimer assembly in both purified and cell culture systems. Aim II. Establish mechanisms for H4B and heme catalytic function, and evaluate candidate protein elements. NOS make NO from Arg in a two-step reaction that forms N-hydroxyArg as an intermediate and requires bound H4B. The principal investigator hypothesizes the NOS heme receives an electron from its reductase domain to bind O2 and initiate either step of NO synthesis H4B then functions by out competing the reductase domain in providing an electron to the heme FE(II)O2 species, and by acting as a 1-electron oxidant in the second step. He will test the proposed roles for heme and H4B in all three NOSs by performing single turnover studies that utilize stopped-flow spectroscopy rapid freeze EPR spectroscopy, and rapid chemical quench methods. Experiments will determine if kinetic or quantitative relationships exist between FeO2 disappearance, H4B radical formation, and product formation in either step of NO synthesis. The principal investigator next will determine how residues that surround H4B or heme affect relationships, to establish how they control H4B and heme function. Aim III. Demonstrate electron transfer between the reductase domain and H4B. If an H4B radical forms during Arg hydroxylation, the reductase domain must furnish an electron to reduce it to H4B before initiating the second step of NO synthesis. He will test this by monitoring buildup and decay of the H4B radical during a single turnover in NADPH-reduced NOS under conditions where the reductase domain either can or cannot provide an electron to H4B. This will involve single-mixing experiments followed by EPR analysis, and double-mixing experiments that temporally separate H4B radical formation from its reduction. AIM IV. Evaluate candidate protein elements in electrons to heme and H4B, and establish mechanisms. During NO synthesis, a NOS oxygenase domain must interact with a partner reductase domain to obtain electrons for its heme and H4B groups. The principal investigator will: (1) Determine if reductase-oxygenase pairing within a dimer is similar in all NOS. (2) Generate truncated NOS to analyze domain interaction by crystallography.(3) Evaluate a role for charged surface residues in domain docking,(4) Evaluate three candidate pathways for electron delivery to heme and/or H4B.